Methods for Detecting and Identifying Genomic Nucleic Acids

ABSTRACT

The present invention relates to a method of identifying a target genomic nucleic acid sequence including hybridizing a set of probes to the target genomic nucleic acid sequence, wherein the set of probes has a unique associated barcode sequence for identification of the target genomic nucleic acid sequence, wherein each probe of the set includes (1) a complementary sequence complementary to a first strand of the target genomic nucleic acid sequence and (2) the associated barcode sequence or a portion of the associated barcode sequence, sequencing the associated barcode sequence from probes hybridized to the target genomic nucleic acid sequence using a fluorescence-based sequencing method, and identifying the target genomic nucleic acid sequence by the sequenced barcode sequence.

RELATED APPLICATION DATA

This application claims priority to U.S. Provisional Application No.62/309,714 filed on Mar. 17, 2016 which is hereby incorporated herein byreference in entirety for all purposes.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under HG005550 awardedby National Institutes of Health. The government has certain rights inthe invention.

FIELD

The present invention relates in general to methods of in situsequencing of barcodes associate with oligonucleotide probes hybridizedto a target genomic nucleic acid. In this manner, methods andcompositions for detecting, sequencing, identifying, measuring,counting, and/or segmenting genomic features in cells are provided.

BACKGROUND

Nucleic acids may be imaged in situ using nucleic acid hybridizationmethods. For genomic imaging in situ, short or long probes modified withfluorophores or other detecting moieties are hybridized to the genome.For example, short oligonucleotide probes are hybridized to the genome,where each probe bears either one or more fluorescent moieties, or oneor more sites for secondary hybridization by a fluorophore-bearingoligonucleotide. However, the multiplexity of this method, e.g. thenumber of distinct genomic loci able to be labeled, is limited to eitherF×N using F spectrally distinct fluorescent moieties to label F genomicloci in each of N cycles of probe hybridization, or is bounded by N×kusing k combinations of fluorescent signals, each comprised of aspecific number and/or combination of the F spectrally distinctfluorophores, referred to as “colorimetric” barcoding (e.g. red+blue asa distinct label, or 2× red vs 1× red if various levels of red can bedistinguished). Unfortunately this number is far fewer than the numberof genomic loci, which meaningfully extends into the millions.Accordingly, methods of multiplexing the detection of a large number ofgenomic loci is needed.

SUMMARY

The present disclosure provides probes including one or more barcodesthat can be used to hybridize to target genomic nucleic acids. Thebarcodes may be sequenced using a fluorescence-based sequencing methodto detect and identify the target genomic nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The foregoing and other features and advantages ofthe present invention will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawing in which:

FIG. 1 is a schematic depicting hybridization of probes includingbarcodes to target genomic nucleic acids.

FIG. 2 is a schematic of a fluorescence based sequencing method used tosequence the barcodes of probes hybridized to target genomic nucleicacids.

FIG. 3 is a schematic of the use of cyclic reversible polymerizationhybridization chain reaction (HCR) for sequencing of barcodes.

FIG. 4 depicts the results of an experiment directed to the sequencingof barcodes hybridized to target genomic nucleic acids.

FIG. 5 is an image of cells with an expandable matrix having undergonein situ genome sequencing. IMR90 cells were stained with Oligopaintstargeting 300 Kb-1 Mb unique regions on each chromosome with 3750Oligopaints per region (˜8 probes/Kb). Cells were then expanded ˜4.5× inExM gel. Oligopaint circularization, followed by Rolling CircleAmplifcation, and 1 round of sequencing by ligation was performed.Various colors represent 1 base of SoliD sequencing. Scale bar=10microns.

FIG. 6A-E are directed to experiments demonstrating that acryditemodified Oligopaints allow tethering of Oligopaint to ExM gel matrix.FIG. 6A is a schematic depicting Oligopaint with Acrydite modification.Acrydite (Yellow trapezoid) is incorporated onto the 5′ end of eachOligopaint. Oligopaints are visualized with a fluorophore labeledsecondary oligo binding to the Mainstreet (non-genomic sequence upstreamof the complementary nucleic acid sequence. FIG. 6B-6E depict PGP1Fcells stained with ˜20,000 Oligopaints (green) targeting 2.1 Mb regionon q arm of Chromosome 19 (9.2 probes/Kb) in ExM gel. Non-modifiedOligopaints are shown in FIGS. 6B-6C. Acrydite modified Oligopaints areshown in FIGS. 6D-6E. Acrydite modified Oligopaints remain attached toExpansion gel after Oligopaint removal by treatment with 70% formamideat 73 degrees (FIG. 6E), while non-modified Oligopaints do not (FIG.6C). Scale bar=10 microns.

DETAILED DESCRIPTION

Terms and symbols of nucleic acid chemistry, biochemistry, genetics, andmolecular biology used herein follow those of standard treatises andtexts in the field, e.g., Komberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, HumanMolecular Genetics, Second Edition (Wiley-Liss, New York, 1999);Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); and the like.

The disclosure provides use of a plurality or set of probes forhybridization with a given target genomic nucleic acid. The probes cancross a cell membrane and/or a nuclear membrane. The plurality or set ofprobes have a unique associated barcode to identify the probes withinthe plurality or set. The disclosure provides use of a plurality of setsof probes, such that a corresponding plurality of target genomic nucleicacids may be detected and identified, such as in a multiplexed manner.As the associated barcode is unique to the target genomic nucleic acid,sequencing the associated barcode using a fluorescence-based sequencingmethod allows one to detect and identify the target genomic nucleicacid. Since a plurality of sets of probes may be used, a plurality oftarget genomic nucleic acids, such as within a genome, may be detectedand identified, such as in a multiplexed manner, where the plurality ofsets probes are contacted with genomic DNA to hybridize to a pluralityof corresponding target nucleic acids, the associated barcodes aresequenced using a fluorescence-based sequencing method, and theplurality of corresponding target nucleic acids are detected andidentified. The disclosure provides the use of fluorescence-basedsequencing methods such as sequencing by synthesis, sequencing byligation, sequencing by hybridization, sequencing by cyclic reversiblepolymerization hybridization chain reaction (HCR) and the like. Sincefluorescence-based sequencing methods are used to sequence an associatedbarcode corresponding to the plurality or set of probes hybridized to agiven target genomic nucleic acid sequence, the fluorescent signal iseffectively amplified by the number of probes hybridized to the giventarget nucleic acid sequence.

The disclosure provides that a barcode is associated with a plurality orset of oligonucleotide probes such that the barcode uniquely identifiesthe plurality or set of oligonucleotide probes and, therefore, thecorresponding target genomic nucleic acid to which the plurality or setof oligonucleotide probes is hybridized. The disclosure provides thatthe barcode is associated with the plurality or set of oligonucleotideprobes to the extent that each probe in the plurality or set includesthe barcode. The disclosure provides that the barcode is associated withthe plurality or set of oligonucleotide probes to the extent that eachprobe in the plurality or set includes a portion of an entire barcode,such that sequencing of the portions attached to the probes results inthe sequencing of the entire barcode. The portions of the entire barcodeneed not be equal portions. The portions of the barcode may overlap andprovide useful redundancy. Providing portions of an entire barcode onprobes also advantageously shortens the length of the probe to moreeasily facilitate hybridization to a target genomic nucleic acid.

The disclosure provides methods where a plurality of oligonucleotideprobes are hybridized to the genome, such that for every subset of thetotal pool of probes, which correspond to individual genomic loci to belabeled and detected, bears a unique identifying barcode sequence.Alternatively, the subset of probes targeting each single locus may beara number of sequences that function as an ensemble barcode. For example,some probes may bear the first segment of the barcode, while others bearthe second segment, and so on. The barcode sequence may be read out bysequencing by ligation, sequencing by synthesis or sequencing byhybridization, where the signal is effectively amplified by the numberof probes at each locus bearing the barcode or particular segment (e.g.base) of the barcode. The barcode sequence may also be read out bysequencing by cyclic reversible polymerization hybridization chainreaction (HCR).

The disclosure provides a method of identifying a target genomic nucleicacid sequence including hybridizing a set of probes to the targetgenomic nucleic acid sequence, wherein the set of probes has a uniqueassociated barcode sequence for identification of the target genomicnucleic acid sequence, wherein each probe of the set includes (1) acomplementary sequence complementary to a first strand or a secondstrand of the target genomic nucleic acid sequence and (2) theassociated barcode sequence or a portion of the associated barcodesequence, sequencing the associated barcode sequence from probeshybridized to the target genomic nucleic acid sequence using afluorescence-based sequencing method, and identifying the target genomicnucleic acid sequence by the sequenced barcode sequence.

The disclosure provides that the fluorescence-based sequencing method issequencing by synthesis and the probe further includes a primingsequence for annealing a sequencing primer, wherein the sequencingprimer is extended by a DNA polymerase using reversible terminatorfluorescently encoded dNTPs to generate a fluorescent signalcorresponding to the barcode.

The disclosure provides that the fluorescence-based sequencing method issequencing by ligation and the probe further includes a priming sequencefor annealing a sequencing primer, wherein the sequencing primer isextended in either the 5′ or 3′ direction by a DNA ligase usingfluorescently encoded oligonucleotides to generate a fluorescent signalcorresponding to the barcode.

The disclosure provides that the fluorescence-based sequencing method issequencing by hybridization and the probe further includes a firstnucleic acid sequence complementary to a labeled oligonucleotide whichhybridizes to the first nucleic acid sequence.

The disclosure provides that the fluorescence-based sequencing method issequencing by cyclic reversible polymerization hybridization chainreaction.

The disclosure provides that a plurality of probes having a portion ofthe associated barcode sequence constitute a complete associated barcodesequence.

The disclosure provides that the complementary sequence has a nucleotidelength of between 5 and 10,000 bases. The disclosure provides that thecomplementary sequence has a nucleotide length of between 15 and 1,000bases. The disclosure provides that the complementary sequence has anucleotide length of between 20 and 80 bases.

The disclosure provides that the probe includes one or more spacersequences separating the complementary sequence from the associatedbarcode sequence or portion of the associate barcode sequence.

The disclosure provides that the probe includes one or more spacersequences comprising a plurality of dT nucleotides separating thecomplementary sequence from the associated barcode sequence or portionof the associate barcode sequence.

The disclosure provides that the probe includes one or more spacersequences separating functional sequences of the barcode including thecomplementary sequence, the associated barcode sequence or portion ofthe associate barcode sequence, and a priming sequence.

The disclosure provides that the probe includes one or more spacersequences comprising a plurality of dT nucleotides separating functionalsequences of the barcode including the complementary sequence, theassociated barcode sequence or portion of the associate barcodesequence, and a priming sequence.

The disclosure provides that the target genomic nucleic acid has alength of between 10 bp and 1,000,000,000 bp. The disclosure providesthat the target genomic nucleic acid has a length of between 20 bp and1,000,000 bp. The disclosure provides that the target genomic nucleicacid has a length of between 100 bp and 1,000,000 bp. The disclosureprovides that the target genomic nucleic acid has a length of between100,000 bp and 1,000,000,000 bp. The disclosure provides that the targetgenomic nucleic acid is a whole genome.

The disclosure provides that the probe includes one or more additionalbarcode sequences for barcoding characteristics of the target genomicnucleic acid.

The disclosure provides that the probes are oligopaints.

The disclosure provides that the probes further include an attachmentmoiety for attachment to a matrix.

The disclosure provides for a method of multiplexing the identificationof a plurality of target genomic nucleic acid sequences within genomicDNA including hybridizing the genomic DNA with a plurality of probe setscorresponding to the plurality of target genomic nucleic acid sequences,wherein each probe set has a unique associated barcode sequence foridentification of the corresponding target genomic nucleic acidsequence, wherein each probe of each probe set includes (1) acomplementary sequence complementary to a first strand of thecorresponding target genomic nucleic acid sequence and (2) theassociated barcode sequence or a portion of the associated barcodesequence, sequencing the associated barcode sequence from probeshybridized to the plurality of target genomic nucleic acid sequencesusing a fluorescence-based sequencing method, and identifying the targetgenomic nucleic acid sequence by the sequenced barcode sequence. Thedisclosure provides that the plurality of target genomic nucleic acidsequences within genomic DNA is between 5 and 100,000,000 target genomicnucleic acid sequences.

Probes

Nucleic acid sequences or oligonucleotide probes according to thepresent disclosure may have any desired length. The term “probe” refersto a single-stranded oligonucleotide sequence that will recognize andform a hydrogen-bonded duplex with a complementary sequence in a targetnucleic acid sequence or its cDNA derivative. The probe includes atarget hybridizing nucleic acid sequence. A probe provided by thedisclosure includes a complementary sequence complementary to a strandof the target genomic nucleic acid sequence and the associated barcodesequence or a portion of the associated barcode sequence. Accordingly,the term “probe” may also be understood as including the barcode or anysequence feature needed for fluorescence-based sequencing of thebarcode. Oligonucleotide or polynucleotide probes may be designed, ifdesired, with the aid of a computer program such as, for example,DNAWorks, or Gene2Oligo.

The complementary sequence may have a nucleotide length between about 15and about 1000 bases. The complementary sequence may have a nucleotidelength between about 15 and about 500 bases. The complementary sequencemay have a nucleotide length between about 15 and about 400 bases. Thecomplementary sequence may have a nucleotide length between about 15 andabout 300 bases. The complementary sequence may have a nucleotide lengthbetween about 15 and about 200 bases. The complementary sequence mayhave a nucleotide length between about 15 and about 100 bases. Thecomplementary sequence may have a nucleotide length between about 15 andabout 90 bases. The complementary sequence may have a nucleotide lengthbetween about 15 and about 80 bases. The complementary sequence may havea nucleotide length between about 15 and about 70 bases. Thecomplementary sequence may have a nucleotide length between about 15 andabout 60 bases. The complementary sequence may have a nucleotide lengthbetween about 15 and about 50 bases. The complementary sequence may havea nucleotide length between about 15 and about 40 bases. Thecomplementary sequence may have a nucleotide length between about 15 andabout 30 bases. The complementary sequence may have a nucleotide lengthbetween about 20 and about 1000 bases. The complementary sequence mayhave a nucleotide length between about 20 and about 500 bases. Thecomplementary sequence may have a nucleotide length between about 20 andabout 100 bases. The complementary sequence may have a nucleotide lengthbetween about 20 and about 80 bases. The complementary sequence may havea nucleotide length between about 20 and about 40 bases. Thecomplementary sequence may have a nucleotide length between about 20 andabout 100 bases. The complementary sequence may have a nucleotide lengthbetween about 20 and about 60 bases. The complementary sequence may havea nucleotide length of about 22, 32, 40, 50 or 60 bases.

The disclosure provides for the optimization of the length of thecomplementary region based on one or more of the following: On-targetvs. off-target thermodynamic specificity, [e.g. such that thethermodynamic penalty of a probe hybridizing off-target vs. on-target,which is related to the difference in free energy between the twohybridization states (off vs on target), is great enough that underparticular hybridization reaction conditions (e.g. salt, formamide,temperature, competing probes, etc., which all effect the equilibriumconstant of the hybridization reaction (A+B< >AB), the hybridizationreaction will cause on-target hybridizations to be maximized whileoff-target hybridizations are minimized]; and hybridization reactionkinetics [e.g. that the reaction will be driven to a sufficient state ofcompletion (1%, 10%, 20%, 50%, 100% of probes hybridized to the target)within a certain amount of reaction time], such that the genomic locuscan be identified.

The disclosure provides probes which may be oligonucleotide orpolynucleotide probes. Such oligonucleotide or polynucleotide probes maybe referred to as Oligopaint probes or Oligopaints or chromosome paintsas is known in the art. See US-2010-0304994 hereby incorporated byreference in its entirety. Oligopaint probes or Oligopaints orchromosome paints are detectable markers that label chromosomes alongtheir entire length, permitting physicians and researchers to identifychromosomes and decipher chromosome rearrangements. The presentdisclosure provides the use of barcode sequences that, when sequenced,serve as detectable markers for the target genomic nucleic acid.

Probes, such as Oligopaints, have a high resolution useful in detectingand identifying target genomic nucleic acids. As used herein, the term“resolution” refers to the ability to distinguish (e.g., label) betweentwo points on a polynucleotide sequence (e.g., two points along thelength of a chromosome). As used herein, the term “high resolution”refers to the ability to detect two or more nucleic acid sequenceshaving a distance of less than 6×10⁶ base pairs apart (e.g., on achromosome). In certain aspects, two or more high resolution Oligopaintshave a resolution of about 500 kilobases apart or fewer, 400 kilobasesapart or fewer, 300 kilobases apart or fewer, 200 kilobases apart orfewer, 100 kilobases apart or fewer, 90 kilobases apart or fewer, 80kilobases apart or fewer, 70 kilobases apart or fewer, 60 kilobasesapart or fewer, 50 kilobases apart or fewer, 40 kilobases apart orfewer, 30 kilobases apart or fewer, 20 kilobases apart or fewer, 19kilobases apart or fewer, 18 kilobases apart or fewer, 17 kilobasesapart or fewer, 16 kilobases apart or fewer, 15 kilobases apart orfewer, 14 kilobases apart or fewer, 13 kilobases apart or fewer, 12kilobases apart or fewer, 11 kilobases apart or fewer, 10 kilobasesapart or fewer, 9 kilobases apart or fewer, 8 kilobases apart or fewer,7 kilobases apart or fewer, 6 kilobases apart or fewer, 5 kilobasesapart or fewer, 4 kilobases apart or fewer, 3 kilobases apart or fewer,2 kilobases apart or fewer or 1 kilobase apart or fewer. In certainaspects, two or more high resolution Oligopaints have a resolution ofabout 1900 bases apart or fewer, 1800 bases apart or fewer, 1700 basesapart or fewer, 1600 bases apart or fewer, 1500 bases apart or fewer,1400 bases apart or fewer, 1300 bases apart or fewer, 1200 bases apartor fewer, 1100 bases apart or fewer, 1000 bases apart or fewer, 900bases apart or fewer, 800 bases apart or fewer, 700 bases apart orfewer, 600 bases apart or fewer, 500 bases apart or fewer, 400 basesapart or fewer, 300 bases apart or fewer, 200 bases apart or fewer, 100bases apart or fewer, 95 bases apart or fewer, 90 bases apart or fewer,85 bases apart or fewer, 80 bases apart or fewer, 75 bases apart orfewer, 70 bases apart or fewer, 65 bases apart or fewer, 60 bases apartor fewer, 55 bases apart or fewer, 50 bases apart or fewer, 45 basesapart or fewer, 40 bases apart or fewer, 35 bases apart or fewer, 30bases apart or fewer, 25 bases apart or fewer, 20 bases apart or fewer,15 bases apart or fewer, 10 bases apart or fewer or down to theindividual base pair. In certain aspects, two or more high resolutionOligopaints have a resolution of between about 10 bases and about 2000bases, between about 10 bases and about 1000 bases, between about 10bases and about 500 bases, between about 15 bases and about 250 bases,between about 15 bases and about 100 bases, between about 20 bases andabout 50 bases, or between about 20 bases and about 30 bases.

As used herein, the term “sensitivity,” with respect to probes, refersto the number of target nucleotide bases (e.g., target genomicnucleotide bases) that are complementary to a particular probe, i.e.,the number of target nucleotide bases to which a particular probe canhybridize (i.e., the smallest band size that can be detected). Incertain aspects, high resolution probes have a resolution of about 1kilobase, about 1900 bases, about 1800 bases, about 1700 bases, about1600 bases apart, about 1500 bases, about 1400 bases, about 1300 bases,about 1200 bases, about 1100 bases, about 1000 bases, about 900 bases,about 800 bases, about 700 bases, about 600 bases, about 500 bases,about 400 bases, about 300 bases, about 200 bases, about 100 bases,about 95 bases, about 90 bases, about 85 bases, about 80 bases, about 75bases, about 70 bases, about 65 bases, about 60 bases, about 55 bases,about 50 bases, about 45 bases, about 40 bases, about 35 bases, about 30bases, about 25 bases, about 20 bases, about 15 bases, about 10 bases,or about 5 bases. In certain aspects, the number of target nucleotidebases that are complementary to a probe are consecutive (e.g.,consecutive genomic nucleotide bases).

The disclosure provides that probes are complementary to genomic nucleicsequences that are present in low or single copy numbers (e.g., genomicnucleic sequences that are not repetitive elements). As used herein, theterm “repetitive element” refers to a DNA sequence that is present inmany identical or similar copies in the genome. Repetitive elements arenot intended to refer to a DNA sequence that is present on each copy ofthe same chromosome (e.g., a DNA sequence that is present only once, butis found on both copies of chromosome 11, would not be considered arepetitive element, and would be considered a sequence that is presentin the genome as one copy). The genome consists of three broad sequencecomponents: Single copy or at least very low copy number DNA(approximately 60% of the human genome); moderately repetitive elements(approximately 30% of the human genome); and highly repetitive elements(approximately 10% of the human genome). For a review, see HumanMolecular Genetics, Chapter 7 (1999), John Wiley & Sons, Inc.

The disclosure provides the use of small probes, such as Oligopaints. Asused herein, the term “small probe” refers to a probe of between about 5bases and about 100 bases long, or a probe of about 5 bases, about 10bases, about 15 bases, about 20 bases, about 25 bases, about 30 bases,about 35 bases, about 40 bases, about 45 bases, about 50 bases, about 55bases, about 60 bases, about 65 bases, about 70 bases, about 75 bases,about 80 bases, about 85 bases, about 90 bases, about 95 bases, or about100 bases. Small probes can access targets that are not accessible tolonger oligonucleotide probes. For example, in certain aspects smallprobes can pass into a cell, can pass into a nucleus, and/or canhybridize with targets that are partially bound by one or more proteins,etc. Small probes are also useful for reducing background, as they canbe more easily washed away than larger hybridized oligonucleotidesequences.

The present disclosure provides for the use of a plurality of probeswith a unique barcode sequence to hybridize to, detect and identify atarget nucleic acid sequence. The plurality of probes may be a set orlibrary of probes with a unique associated barcode which uniquelyidentifies the library or set of probes, and therefore, the targetgenomic sequence to which they are hybridized. The library may containmultiple probes with sequence complementarity to a single genomic locus,where the genomic locus has sequence variation including substitutions,mutations, deletions, or insertions of bases, such that under certainhybridization conditions the probability of all probes binding to alocus exhibiting sequence variation is not equal, and is biased in favorof binding one probe containing the corresponding and complementarysequence variation or is capable of encoding the sequence variation andwhere specificity may be gained by competition between a set of suchprobes. The associated barcode may be in the form of a portion of theentire barcode on one or more probes, such that the total barcodesequence may be determined by sequencing the portions of the barcodesequence. The portions may be overlapping or nonoverlapping.

The disclosure provides the use of one or more spacer sequences orregions that separate functional regions of the probe, where thefunctional regions are defined as those involved in nucleic acidhybridization, signal amplification, or sequencing. Spacer sequences aredesigned to specifically not interact with itself, e.g. byintramolecular hybridization to form hairpins or other DNA secondarystructures, or the target genomic sequence or other sequences with whichit may contact, such as intermolecular hybridization to other probes,RNA molecules, or other genomic loci, or to not promote intermolecularaffinity to other biomolecular features such as proteins. Exemplaryspacer sequences are poly-T sequences including d(T)₄, d(T)₆, and d(T)₈.

The disclosure provides the design and use of multiple probes thathybridize to a target genomic locus to create a combined signal whichcan be used to detect and identify the target genomic locus. As anexample, a plurality or set or library of DNA oligonucleotide probes aredesigned such that a number of DNA oligonucleotide probes bearing asingle barcode are used to hybridize to a genomic locus, such that whensequenced the set of probes generates a combined signal with enhancedphoton yield and signal-to-noise ratio.

Barcodes and Other Sequences for Use with Probes

The present disclosure provides for the use of barcode sequences todifferentially label sets of probes from one another. A barcode sequenceis designed to be unique to the set of probes so the barcode can be usedto detect and identify the target genomic sequence by sequencing thebarcode or portions of the barcode from the probes hybridized to thetarget genomic sequence. The barcodes are designed such that they do nothybridize to the target nucleic acid sequence. Barcode sequences may beoligonucleotide sequences between 4 and 40 nucleotides. Barcodesequences may be oligonucleotide sequences between 8 and 25 nucleotides.Barcode sequences may be oligonucleotide sequences between 8 and 20nucleotides.

The disclosure provides that the barcode may include any desired numberof nucleotides sufficient to identify a corresponding desired number oftarget genomic nucleic acid sequences. A barcode is aninformation-theoretical construct that yields an ordered sequence offluorescent signals when interrogated over time. The architecture of thebarcode is dependent on the sequencing method used. For example, usingcyclic HCR, the interrogation method is sequencing by hybridization(SBH), and a typical hybridization reaction is capable of distinguishingbetween regions of sequence ˜20-25 bp in length per ordered signal.Therefore, as an example, a barcode composed of 20 ordered signals wouldrequire, 20×25 nucleotides of “barcode sequence” on the probe set,library or pool (e.g. distributed among the probes targeting a singlelocus). Other exemplary SBH interrogation methods (e.g. oligoPAINT) uses8-bp motifs, so a barcode composed of 20 ordered signals would includeat least 8×20 nucleotides. For sequencing by synthesis and ligation,detection of individual nucleotides can serve as the readout of orderedsignals, so therefore a barcode composed of 20 ordered signals could becontained in 1×20 nucleotides. For sequencing by ligation, detection of5-nt motifs can serve as the readout of ordered signals, so a barcodecomposed of 20 ordered signals could be contained in 5×20 nucleotides.In this way, barcode sequences are always composed of nucleotidesequences, but the relationship between the length of the “barcode” andthe length of the sequence representing the barcode may be far from 1:1(e.g. 1:25 in the case of SBH for cyclic HCR).

The probes are designed such that they include features that allow thebarcodes be sequenced using fluorescence-based sequencing methods suchas sequencing by synthesis, sequencing by ligation, sequencing byhybridization or sequencing by cyclic reversible polymerizationhybridization chain reaction as is known in the art.

For example, for sequencing by synthesis, each barcoding region containsa priming region, where a sequencing primer may be annealed to form apartially double-stranded region of DNA, and an arbitrary number ofbases serving as a specific barcode that is used to identify the genomicloci, such that extension of the sequencing primer by a DNA polymeraseusing reversible-terminator fluorescently encoded dNTPs will generate afluorescent signal corresponding to the barcode, i.e. the probe mayfurther include a priming sequence for annealing a sequencing primer,wherein the sequencing primer is extended by a DNA polymerase usingreversible terminator fluorescently encoded dNTPs to generate afluorescent signal corresponding to the barcode.

For example, for sequencing by ligation, each barcoding region containsa priming region, where a sequencing primer may be annealed to form apartially double-stranded region of DNA, and an arbitrary number ofbases serving as a specific barcode that is used to identify the genomicloci, such that extension of the sequencing primer in either the 5′ or3′ direction by a DNA ligase using fluorescently encodedoligonucleotides will generate a fluorescent signal corresponding to thebarcode, i.e. the probe may further include a priming sequence forannealing a sequencing primer, wherein the sequencing primer is extendedin either the 5′ or 3′ direction by a DNA ligase using fluorescentlyencoded oligonucleotides to generate a fluorescent signal correspondingto the barcode.

For example, for sequencing by hybridization, each barcoding regioncontains one or more regions of DNA sequence which may be probed usingcomplementary fluorescently labeled oligonucleotides by nucleic acidhybridization, including where the nucleic acid hybridization is by DNAPAINT, e.g. where the fluorescently labeled detecting oligos are notstably hybridized to the genomically-targeted probe, but rather exist inequilibrium state such that super-resolution microscopy is achieved bytemporal separation of molecules beyond the diffraction limit bystochastic blinking, photon accumulation, and Gaussian fitting todetermine the localization of the detecting probe.

The disclosure provides for the use of standard Next Generationsequencing chemistries and deep three dimensional imaging for highthroughput information readout for sequencing of the barcodes describedherein. The Next Generation sequencing chemistries that utilizefluorescence imaging include ABI SoLiD (Life Technologies), in which asequencing primer on a template is ligated to a library of fluorescentlylabeled nonamers with a cleavable terminator. After ligation, the beadsare then imaged using four color channels (FITC, Cy3, Texas Red andCy5). The terminator is then cleaved off leaving a free-end to engage inthe next ligation-extension cycle. After all dinucleotide combinationshave been determined, the images are mapped to the color code space todetermine the specific base calls per template. The workflow is achievedusing an automated fluidics and imaging device (i.e. SoLiD 5500 W GenomeAnalyzer, ABI Life Technologies). Another sequencing platform usessequencing by synthesis, in which a pool of single nucleotide with acleavable terminator is incorporated using DNA polymerase. Afterimaging, the terminator is cleaved and the cycle is repeated. Thefluorescence images are then analyzed to call bases for each DNAamplicons within the flow cell (HiSeq, Illumia). General sequencingmethods known in the art, such as sequencing by extension withreversible terminators, fluorescent in situ sequencing (FISSEQ),pyrosequencing, massively parallel signature sequencing (MPSS) and thelike (described in Shendure et al. (2004) Nat. Rev. 5:335, incorporatedherein by reference in its entirety), are suitable for use in thepresent methods. Reversible termination methods use step-wisesequencing-by-synthesis biochemistry that coupled with reversibletermination and removable fluorescence (Shendure et al. supra and U.S.Pat. Nos. 5,750,341 and 6,306,597, incorporated herein by reference.)FISSEQ is a method useful for barcode sequencing whereby DNA is extendedby adding a single type of fluorescently-labelled nucleotidetriphosphate to the reaction, washing away unincorporated nucleotide,detecting incorporation of the nucleotide by measuring fluorescence, andrepeating the cycle. At each cycle, the fluorescence from previouscycles is bleached or digitally subtracted or the fluorophore is cleavedfrom the nucleotide and washed away. FISSEQ is described further inMitra et al. (2003) Anal. Biochem. 320:55, incorporated herein byreference in its entirety for all purposes. According to certainaspects, the barcodes can be interrogated using methods known to thoseof skill in the art including fluorescently labeledoligonucleotide/DNA/RNA hybridization, primer extension with labeledddNTP, sequencing by ligation (i.e. ABI SoLiD) and sequencing bysynthesis (i.e. Illumina). Ligated circular padlock probes described inLarsson, et al., (2004), Nat. Methods 1:227-232 can be used to detectmultiple sequence targets in parallel, followed by eithersequencing-by-ligation, -synthesis or -hybridization of the barcodesequences in the padlock probe to identify individual targets.

The disclosure provides that the probe include an entire barcodesequence. The disclosure provides that the probe include a portion of abarcode sequence, such as where the entire barcode encoding the identityof the genomic loci is distributed among the multiple probes targeting asingle genomic loci, such that each probe contains only a subset of theinformation in the full barcode, but in a manner such that the barcodemay still be read out programmatically. As an example half the probestargeting a genomic locus contain five values of the barcode, sequencingof which is initiated using a particular sequencing primer, while theother half of the probes targeting the same genomic locus containanother five values of the barcode, sequencing of which is initiatedusing a second sequencing primer, enabling a ten-value barcode to bedetermined. As an example, each probe bears one segment of a barcode,which are read out sequentially in a deterministic order by cyclicreversible hybridization chain reaction (HCR).

The disclosure provides for the probe or the barcode to include errordetection and/or error correction features. Error detection andcorrection methods suitable for use in the present disclosure aredescribed at woorld wide websiteen.wikipedia.org/wiki/Error_detection_and_correction. For example, thebarcode is constructed such that additional information is encodedbeyond that necessary for identification. Exemplary additionalinformation includes one or more sequences to effectively increase theHamming distance between the encoded strings (e.g. constructing barcodein a barcode space of 4⁵⁰ where only 4²⁰ barcodes are needed to identifyeach genomic locus). For example, if a barcode is composed of 20 ordereddetections of 4 identities (4{circumflex over ( )}20 combinations),which is used to identify 4{circumflex over ( )}20 items, when a singleerror occurs in detecting the barcode [either 1) a missed signal or 2)an incorrect signal at any point in the barcode], one would either 1) beunable to distinguish between 4 possible items it could be or 2) couldbe mis-identify the item, because all 4{circumflex over ( )}20 barcodescorrespond to items to be detected. If one uses 50 ordered detections of4 identities (4{circumflex over ( )}50) to identify the same4{circumflex over ( )}20 items, if you have an error as above, it isunlikely that the erroneous barcode will correspond to any of the4{circumflex over ( )}20 real barcodes. Therefore, which real barcode isclosest to the erroneous detected barcode in hamming distance isidentified, and it is most likely to actually be that barcode. In thisway, the method includes both detecting an error and correcting anerror.

A probe or barcode may be constructed to incorporate error detectionand/or correction codes such as parity bits, checksums, Golay encoding,or any other method for detecting and/or correcting errors. The presentdisclosure contemplates use of redundancy for error detection andcorrection. Barcode sequences are constructed such that they are twoexact repeats of a smaller identifying label. When there is an error indetecting a certain signal, (missing signal—e.g. fluorescence is notidentified) but is detected later in the redundant part of the barcode,at that time the signal is detected. The barcode can be accuratelyidentified using a redundancy in the barcode sequence. The presentdisclosure contemplates use of Golay encoding for error detection andcorrection. For a binary ID/label of length 12 (12 ordered detections of2 identities), on the actual probes, a longer barcode is used composedof 24 ordered detections of 2 identities (2{circumflex over ( )}24) thatis the binary Golay representation of the length-12 label, whichcontains additional ordered detections composed according to the Golayalgorithm G24 (see world wide websiteen.wikipedia.org/wiki/Binary_Golay_code). When detecting the barcode, 24ordered detections of 2 identities are read out, (Golay24 represents abinary string of length 12 using 24 bits), and then any 3 individualincorrect detection events can be corrected, or an error is detectedfrom up to any 7 individual incorrect detection events, of the barcodecomposed of 24 detection events. An additional sequence or sequences maybe included in the probe or barcode as needed to address other sourcesof error including if information is lost (e.g. hybridization is lessthan 100% complete, causing some subset of the probes to be missing atthe genomic locus) or erroneous (e.g. a hybridization event isnon-specific, generating a potential false-positive). For example, theentire barcode is divided among probes, such as when a 12-bit label isconstructed into a 24 bit Golay24 barcode (barcode consisting of 24ordered detections of 2 identities), and the 24-bit barcode isdistributed among 24 probes targeting a locus such that each probe bears1 part of the barcode. When hybridization is only 90% efficient, suchthat on average only 21 of the 24 probes make it to the target locus,using Golay encoding, up to 3 incorrect bits in a 12 bit label can becorrected using G24 encoding. Accordingly, missing 3 random bits of this24 bit barcode does not prevent identification by the barcode.

Errors can be detected and/or corrected by using additional informationthat results from sequencing. For example, the encoded information inthe barcode plus additional information for error correction/detectionmay be divided among multiple probes such that a single probe in theabsence of the other probes may be identified as an error, or so thatthe information may still be reconstructed even with some probesmissing. Additional cross-probe information may also be encoded, e.g.signal from two probes could be known to be free of error if each probeencodes information about the other probe.

The disclosure provides for a number of DNA oligonucleotide probes in aset to include one or more barcode regions used for identification ofthe genomic locus. The disclosure provides for a subset of probes in theset to contain additional barcoded information used to convey sequencevariation, e.g. a probe containing complementary sequence to a genomicvariation contains extra bases of the barcode or additional barcodingregions used to convey this additional sequence-variation information inaddition to the information about the coordinates of the genomic locusbearing the particular locus barcode.

Target Genomic Sequence

The disclosure provides for the hybridization of probes to a targetnucleic acid sequence, such as a target genomic nucleic acid sequence,where the probes have a unique associated barcode indicating the targetgenomic sequence. The target genomic nucleic acid sequence may be agenomic locus. The size of the genomic locus identified by a uniqueassociated barcode may be between 100 bp and the whole genome. Exemplarylengths include that of a single histone (about 100-200 bp), a singlegene (about 1-3 kb), a 1-2 Mb region of the genome, an arm of achromosome (100 to 600 Mb) a single chromosome (100-1000 Mb), a wholegenome (on the order of 1-2 Gb) (such as for distinguishing betweenwhole bacterial genomes).

The disclosure provides that the target nucleic acid sequence may be agenomic nucleic acid sequence or region of a genomic nucleic acid, suchas a chromosome or a sub-chromosomal region. The probes described hereincan be used to detect and identify chromosomes and sub-chromosomalregions of chromosomes during various phases of the cell cycleincluding, but not limited to, interphase, preprophase, prophase,prometaphase, metaphase, anaphase, telophase and cytokenesis.

As used herein, the term “chromosome” refers to the support for thegenes carrying heredity in a living cell, including DNA, protein, RNAand other associated factors. The conventional international system foridentifying and numbering the chromosomes of the human genome is usedherein. The size of an individual chromosome may vary within amulti-chromosomal genome and from one genome to another. A chromosomecan be obtained from any species. A chromosome can be obtained from anadult subject, a juvenile subject, an infant subject, from an unbornsubject (e.g., from a fetus, e.g., via prenatal test such asamniocentesis, chorionic villus sampling, and the like or directly fromthe fetus, e.g., during a fetal surgery) from a biological sample (e.g.,a biological tissue, fluid or cells (e.g., sputum, blood, blood cells,tissue or fine needle biopsy samples, urine, cerebrospinal fluid,peritoneal fluid, and pleural fluid, or cells therefrom) or from a cellculture sample (e.g., primary cells, immortalized cells, partiallyimmortalized cells or the like). In certain exemplary embodiments, oneor more chromosomes can be obtained from one or more genera including,but not limited to, Homo, Drosophila, Caenorhabiditis, Danio, Cyprinus,Equus, Canis, Ovis, Ocorynchus, Salmo, Bos, Sus, Gallus, Solanum,Triticum, Oryza, Zea, Hordeum, Musa, Avena, Populus, Brassica, Saccharumand the like.

As used herein, the term “chromosome banding” refers to differentialstaining of chromosomes resulting in a pattern of transverse bands ofdistinguishable (e.g., differently or alternately colored) regions, thatis characteristic for the individual chromosome or chromosome region(i.e., the “banding pattern”). Conventional banding techniques includeG-banding (Giemsa stain), Q-banding (Quinacrine mustard stain),R-banding (reverse-Giemsa), and C-banding (centromere banding). Thedisclosure provides for the use of barcodes to achieve chromosomebanding.

As used herein, the term “karyotype” refers to the chromosomecharacteristics of an individual cell, cell line or genome of a givenspecies, as defined by both the number and morphology of thechromosomes. Karyotype can refer to a variety of chromosomalrearrangements including, but not limited to, translocations,insertional translocations, inversions, deletions, duplications,transpositions, anueploidies, complex rearrangements, telomere loss andthe like. Typically, the karyotype is presented as a systematized arrayof prophase or metaphase (or otherwise condensed) chromosomes from aphotomicrograph or computer-generated image. Interphase chromosomes mayalso be examined. The disclosure provides for the use of barcodes toachieve karotyping.

As used herein, the terms “chromosomal aberration” or “chromosomeabnormality” refer to a deviation between the structure of the subjectchromosome or karyotype and a normal (i.e., non-aberrant) homologouschromosome or karyotype. The deviation may be of a single base pair orof many base pairs. The terms “normal” or “non-aberrant,” when referringto chromosomes or karyotypes, refer to the karyotype or banding patternfound in healthy individuals of a particular species and gender.Chromosome abnormalities can be numerical or structural in nature, andinclude, but are not limited to, aneuploidy, polyploidy, inversion,translocation, deletion, duplication and the like. Chromosomeabnormalities may be correlated with the presence of a pathologicalcondition or with a predisposition to developing a pathologicalcondition. Chromosome aberrations and/or abnormalities can also refer tochanges that are not associated with a disease, disorder and/or aphenotypic change. Such aberrations and/or abnormalities can be rare orpresent at a low frequency (e.g., a few percent of the population (e.g.,polymorphic)). The disclosure provides for the use of barcodes toidentify “chromosomal aberration” or “chromosome abnormality”.

The disclosure provides that the target genomic nucleic acid sequence,such as DNA, can be inside the cell or on a substrate, such as glass,for example as by a “metaphase spread” technique where chromosomes arearrayed on a slide, which is common for karyotyping. The DNA could be ina natural or artificial conformation, e.g. stretched within a flow cell.

Making Probes

Probes described herein, whether a “chromosome paint” or an “Oligopaint”refers to polynucleotides have sequences complementary to anoligonucleotide sequence, e.g., a portion of a DNA sequence e.g., aparticular chromosome or sub-chromosomal region of a particularchromosome. The term “probe” may also be understood as including thebarcode or any sequence feature needed for fluorescence-based sequencingof the barcode.

In general, a plurality or set or library of nucleic acid probes, suchas DNA oligonucleotides, may be synthesized using a DNA microarray, or aDNA chip. The oligonucleotides may contain one or more sequences usedfor the purpose of amplification by polymerase chain reaction (PCR), invitro transcription (IVT), and other biochemical processing steps suchas adding additional sequence by ligation or polymerization,single-stranding, and processing by restriction enzymes, in order togenerate a final library of oligonucleotides. Such methods are known tothose of skill in the art and are described in U.S. Pat. No. 9,476,089,US 2012-0295801 and US 2010-0304994 each of which are herebyincorporated by reference in its entirety.

Probes may be generated from synthetic probes and arrays that are,optionally, computationally patterned (rather than using natural DNAsequences and/or chromosomes as a template). Probes may be made by anysuitable method including array based methods as described in US2010-0304994. Such a method includes the steps of providing at least onesolid support having a plurality of synthetic, single strandedoligonucleotide sequences attached thereto which may nor may not includea barcode sequence or portion of a barcode sequence, wherein a portionof each of the plurality of synthetic, single stranded oligonucleotidesequences is complementary to a portion of a specific chromosomesequence, synthesizing a plurality of complementary strands, each ofwhich is complementary to a synthetic, single stranded oligonucleotidesequence attached to the at least one solid support, removing theplurality of complementary strands from the at least one solid support,amplifying the plurality of complementary strands, and optionallybarcoding the plurality of complementary strands if needed to produce aset of barcoded oligonucleotide paints. Probes, such as Oligopaints oroligonucleotide paints, have a resolution of about two kilobases orfewer. In certain aspects, each probe has a resolution of about onekilobase or fewer or 100 bases or fewer. In certain aspects, the set ofprobes has a resolution of between about 20 bases and about 30 bases.

The disclosure provides that synthesis of oligonucleotide probes (e.g.,Oligopaints) and/or amplification of oligonucleotide probes (e.g.,Oligopaints) can be performed using a support. As used herein, the term“oligonucleotide” is intended to include, but is not limited to, asingle-stranded DNA or RNA molecule, typically prepared by syntheticmeans. Nucleotides of the present invention will typically be thenaturally-occurring nucleotides such as nucleotides derived fromadenosine, guanosine, uridine, cytidine and thymidine. In certainaspects, multiple supports (tens, hundreds, thousands or more) may beutilized (e.g., synthesized, amplified, hybridized or the like) inparallel. Suitable supports include, but are not limited to, slides(e.g., microscope slides), beads, chips, particles, strands, gels,sheets, tubing (e.g., microfuge tubes, test tubes, cuvettes), spheres,containers, capillaries, microfibers, pads, slices, films, plates (e.g.,multi-well plates), microfluidic supports (e.g., microarray chips, flowchannel plates, biochips and the like) and the like. In variousembodiments, the solid supports may be biological, nonbiological,organic, inorganic or combinations thereof. When using supports that aresubstantially planar, the support may be physically separated intoregions, for example, with trenches, grooves, wells, or chemicalbarriers (e.g., lacking a lipid-binding coating). In exemplaryembodiments, supports can be made of a variety of materials including,but not limited to glass, quartz, ceramic, plastic, polystyrene,methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose,nylon and the like and any combination thereof. Such supports and theiruses are well known in the art.

Oligonucleotide sequences useful as probes may be prepared by anysuitable method, e.g., the phosphoramidite method described by Beaucageand Carruthers ((1981) Tetrahedron Lett. 22: 1859) or the triestermethod according to Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185),both incorporated herein by reference in their entirety for allpurposes, or by other chemical methods using either a commercialautomated oligonucleotide synthesizer or high-throughput, high-densityarray methods described herein and known in the art (see U.S. Pat. Nos.5,602,244, 5,574,146, 5,554,744, 5,428,148, 5,264,566, 5,141,813,5,959,463, 4,861,571 and 4,659,774, incorporated herein by reference inits entirety for all purposes). Pre-synthesized oligonucleotides andchips containing oligonucleotides may also be obtained commercially froma variety of vendors.

In an exemplary embodiment, construction and/or selectionoligonucleotides may be synthesized on a solid support using masklessarray synthesizer (MAS). Maskless array synthesizers are described, forexample, in PCT application No. WO 99/42813 and in corresponding U.S.Pat. No. 6,375,903. Other examples are known of maskless instrumentswhich can fabricate a custom DNA microarray in which each of thefeatures in the array has a single stranded DNA molecule of desiredsequence. An exemplary type of instrument is the type shown in FIG. 5 ofU.S. Pat. No. 6,375,903, based on the use of reflective optics. It is adesirable that this type of maskless array synthesizer is under softwarecontrol. Since the entire process of microarray synthesis can beaccomplished in only a few hours, and since suitable software permitsthe desired DNA sequences to be altered at will, this class of devicemakes it possible to fabricate microarrays including DNA segments ofdifferent sequence every day or even multiple times per day on oneinstrument. The differences in DNA sequence of the DNA segments in themicroarray can also be slight or dramatic, it makes no difference to theprocess. The MAS instrument may be used in the form it would normally beused to make microarrays for hybridization experiments, but it may alsobe adapted to have features specifically adapted for the compositions,methods, and systems described herein. For example, it may be desirableto substitute a coherent light source, i.e., a laser, for the lightsource shown in FIG. 5 of the above-mentioned U.S. Pat. No. 6,375,903.If a laser is used as the light source, a beam expanded and scatterplate may be used after the laser to transform the narrow light beamfrom the laser into a broader light source to illuminate the micromirrorarrays used in the maskless array synthesizer. It is also envisionedthat changes may be made to the flow cell in which the microarray issynthesized. In particular, it is envisioned that the flow cell can becompartmentalized, with linear rows of array elements being in fluidcommunication with each other by a common fluid channel, but eachchannel being separated from adjacent channels associated withneighboring rows of array elements. During microarray synthesis, thechannels all receive the same fluids at the same time. After the DNAsegments are separated from the substrate, the channels serve to permitthe DNA segments from the row of array elements to congregate with eachother and begin to self-assemble by hybridization.

Other methods for synthesizing oligonucleotide probes (e.g.,Oligopaints) include, for example, light-directed methods utilizingmasks, flow channel methods, spotting methods, pin-based methods, andmethods utilizing multiple supports.

Light directed methods utilizing masks (e.g., VLSIPS™ methods) for thesynthesis of oligonucleotide probes is described, for example, in U.S.Pat. Nos. 5,143,854, 5,510,270 and 5,527,681. These methods involveactivating predefined regions of a solid support and then contacting thesupport with a preselected monomer solution. Selected regions can beactivated by irradiation with a light source through a mask much in themanner of photolithography techniques used in integrated circuitfabrication. Other regions of the support remain inactive becauseillumination is blocked by the mask and they remain chemicallyprotected. Thus, a light pattern defines which regions of the supportreact with a given monomer. By repeatedly activating different sets ofpredefined regions and contacting different monomer solutions with thesupport, a diverse array of polymers is produced on the support. Othersteps, such as washing unreacted monomer solution from the support, canbe used as necessary. Other applicable methods include mechanicaltechniques such as those described in U.S. Pat. No. 5,384,261.

Additional methods applicable to synthesis and/or amplification ofoligonucleotide probes (e.g., Oligopaints) on a single support aredescribed, for example, in U.S. Pat. No. 5,384,261. For example reagentsmay be delivered to the support by either (1) flowing within a channeldefined on predefined regions or (2) “spotting” on predefined regions.Other approaches, as well as combinations of spotting and flowing, maybe employed as well. In each instance, certain activated regions of thesupport are mechanically separated from other regions when the monomersolutions are delivered to the various reaction sites.

Flow channel methods involve, for example, microfluidic systems tocontrol synthesis of oligonucleotides on a solid support. For example,diverse polymer sequences may be synthesized at selected regions of asolid support by forming flow channels on a surface of the supportthrough which appropriate reagents flow or in which appropriate reagentsare placed. One of skill in the art will recognize that there arealternative methods of forming channels or otherwise protecting aportion of the surface of the support. For example, a protective coatingsuch as a hydrophilic or hydrophobic coating (depending upon the natureof the solvent) is utilized over portions of the support to beprotected, sometimes in combination with materials that facilitatewetting by the reactant solution in other regions. In this manner, theflowing solutions are further prevented from passing outside of theirdesignated flow paths.

Spotting methods for preparation of oligonucleotides on a solid supportinvolve delivering reactants in relatively small quantities by directlydepositing them in selected regions. In some steps, the entire supportsurface can be sprayed or otherwise coated with a solution, if it ismore efficient to do so. Precisely measured aliquots of monomersolutions may be deposited dropwise by a dispenser that moves fromregion to region. Typical dispensers include a micropipette to deliverthe monomer solution to the support and a robotic system to control theposition of the micropipette with respect to the support, or an ink-jetprinter. In other embodiments, the dispenser includes a series of tubes,a manifold, an array of pipettes, or the like so that various reagentscan be delivered to the reaction regions simultaneously.

Pin-based methods for synthesis of oligonucleotide probes on a solidsupport are described, for example, in U.S. Pat. No. 5,288,514.Pin-based methods utilize a support having a plurality of pins or otherextensions. The pins are each inserted simultaneously into individualreagent containers in a tray. An array of 96 pins is commonly utilizedwith a 96-container tray, such as a 96-well microtitre dish. Each trayis filled with a particular reagent for coupling in a particularchemical reaction on an individual pin. Accordingly, the trays willoften contain different reagents. Since the chemical reactions have beenoptimized such that each of the reactions can be performed under arelatively similar set of reaction conditions, it becomes possible toconduct multiple chemical coupling steps simultaneously.

In yet another embodiment, a plurality of oligonucleotide probes (e.g.,Oligopaints) may be synthesized on multiple supports. One example is abead based synthesis method which is described, for example, in U.S.Pat. Nos. 5,770,358, 5,639,603, and 5,541,061. For the synthesis ofmolecules such as oligonucleotides on beads, a large plurality of beadsare suspended in a suitable carrier (such as water) in a container. Thebeads are provided with optional spacer molecules having an active siteto which is complexed, optionally, a protecting group. At each step ofthe synthesis, the beads are divided for coupling into a plurality ofcontainers. After the nascent oligonucleotide chains are deprotected, adifferent monomer solution is added to each container, so that on allbeads in a given container, the same nucleotide addition reactionoccurs. The beads are then washed of excess reagents, pooled in a singlecontainer, mixed and re-distributed into another plurality of containersin preparation for the next round of synthesis. It should be noted thatby virtue of the large number of beads utilized at the outset, therewill similarly be a large number of beads randomly dispersed in thecontainer, each having a unique oligonucleotide sequence synthesized ona surface thereof after numerous rounds of randomized addition of bases.An individual bead may be tagged with a sequence which is unique to thedouble-stranded oligonucleotide thereon, to allow for identificationduring use.

In certain embodiments, a plurality of oligonucleotide probes (e.g.,Oligopaints) may be synthesized, amplified and/or used in conjunctionwith beads and/or bead-based arrays. As used herein, the term “bead”refers to a discrete particle that may be spherical (e.g., microspheres)or have an irregular shape. Beads may be as small as approximately 0.1μm in diameter or as large approximately several millimeters indiameter. Beads typically range in size from approximately 0.1 μm to 200μm in diameter. Beads may comprise a variety of materials including, butnot limited to, paramagnetic materials, ceramic, plastic, glass,polystyrene, methylstyrene, acrylic polymers, titanium, latex,sepharose, cellulose, nylon and the like. In certain aspects, beads mayhave functional groups on their surface which can be used tooligonucleotides (e.g., Oligopaints) to the bead. Oligonucleotidesequences can be attached to a bead by hybridization (e.g., binding to apolymer), covalent attachment, magnetic attachment, affinity attachmentand the like. For example, the bead can be coated with streptavidin andthe nucleic acid sequence can include a biotin moiety. The biotin iscapable of binding streptavidin on the bead, thus attaching the nucleicacid sequence to the bead. Beads coated with streptavidin, oligo-dT, andhistidine tag binding substrate are commercially available (DynalBiotech, Brown Deer, Wis.). Beads may also be functionalized using, forexample, solid-phase chemistries known in the art, such as those forgenerating nucleic acid arrays, such as carboxyl, amino, and hydroxylgroups, or functionalized silicon compounds (see, for example, U.S. Pat.No. 5,919,523).

Various exemplary protecting groups useful for synthesis ofoligonucleotide probes on a solid support are described in, for example,Atherton et al., 1989, Solid Phase Peptide Synthesis, IRL Press. Invarious embodiments, the methods described herein utilize solid supportsfor immobilization of nucleic acids. For example, oligonucleotides maybe synthesized on one or more solid supports. Exemplary solid supportsinclude, for example, slides, beads, chips, particles, strands, gels,sheets, tubing, spheres, containers, capillaries, pads, slices, films,or plates. In various embodiments, the solid supports may be biological,nonbiological, organic, inorganic, or combinations thereof. When usingsupports that are substantially planar, the support may be physicallyseparated into regions, for example, with trenches, grooves, wells, orchemical barriers (e.g., hydrophobic coatings, etc.). Supports that aretransparent to light are useful when the assay involves opticaldetection (see e.g., U.S. Pat. No. 5,545,531). The surface of the solidsupport will typically contain reactive groups, such as carboxyl, amino,and hydroxyl or may be coated with functionalized silicon compounds (seee.g., U.S. Pat. No. 5,919,523).

In one embodiment, the oligonucleotide probes synthesized on the solidsupport may be used as a template for the production of oligonucleotideprobes, such as Oligopaints. For example, the support boundoligonucleotides may be contacted with primers that hybridize to theoligonucleotides under conditions that permit chain extension of theprimers. The support bound duplexes may then be denatured, pooled andsubjected to further rounds of amplification to produce probes, such asOligopaints, in solution. In another embodiment, the support-boundoligonucleotide probes may be removed from the solid, pooled andamplified to produce probes, i.e. Oligopaints, in solution. Theoligonucleotides may be removed from the solid support, for example, byexposure to conditions such as acid, base, oxidation, reduction, heat,light, metal ion catalysis, displacement or elimination chemistry, or byenzymatic cleavage.

In various embodiments, the methods disclosed herein compriseamplification of oligonucleotide sequences, i.e., probes, includingOligopaints. Amplification methods may comprise contacting a nucleicacid with one or more primers that specifically hybridize to the nucleicacid under conditions that facilitate hybridization and chain extension.Exemplary methods for amplifying nucleic acids include the polymerasechain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb.Symp. Quant. Biol. 51 Pt 1:263 and Cleary et al. (2004) Nature Methods1:241; and U.S. Pat. Nos. 4,683,195 and 4,683,202), anchor PCR, RACEPCR, ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988)Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci.U.S.A. 91:360-364), self sustained sequence replication (Guatelli et al.(1990) Proc. Natl. Acad. Sci. U.S.A. 87:1874), transcriptionalamplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:1173), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6:1197),recursive PCR (Jaffe et al. (2000) J. Biol. Chem. 275:2619; and Williamset al. (2002) J. Biol. Chem. 277:7790), the amplification methodsdescribed in U.S. Pat. Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797,6,124,090 and 5,612,199, or any other nucleic acid amplification methodusing techniques well known to those of skill in the art. In exemplaryembodiments, the methods disclosed herein utilize PCR amplification. Thedisclosure provides for the amplification of probes by in vitrotranscription from a promoter to generate an amplified ssRNA pool,followed by reverse transcription to generate a ssDNA library usingmaterials and methods known to those of skill in the art.

In general, high resolution oligonucleotide paints may be made bycomputationally determining genomic spacing of a plurality of synthetic,oligonucleotide sequences, wherein each of the plurality iscomplementary to a portion of a specific chromosome sequence,synthesizing the plurality of synthetic oligonucleotide sequences, andlabelling the plurality of synthetic oligonucleotide sequences with abarcode or portion of an entire barcode to produce a plurality ofoligonucleotide paints, wherein the set of oligonucleotide paints has aresolution of about two kilobases or fewer, and wherein each of aplurality of the oligonucleotide paints is complementary to a targetnucleic acid sequence (e.g., a genomic sequence), such as of 40consecutive nucleotide bases or fewer. Certain exemplary embodiments aredirected to the use of computer software to automate design and/orinterpretation of genomic spacings, complementary sequences and barcodesequences for each specific set of oligonucleotides or Oligopaints. Suchsoftware may be used in conjunction with individuals performinginterpretation by hand or in a semi-automated fashion or combined withan automated system. In at least some embodiments, the design and/orinterpretation software is implemented in a program written in the JAVAprogramming language. The program may be compiled into an executablethat may then be run from a command prompt in the WINDOWS XP operatingsystem. Unless specifically set forth in the claims, the invention isnot limited to implementation using a specific programming language,operating system environment or hardware platform.

Probes Including Attachment Moieties

The disclosure provides probes that include a moiety for attachment to amatrix material to immobilize the probe within or on the matrixmaterial, such as a 3D matrix, for purposes of amplification orsequencing. An exemplary attachment moiety that can be attached toprobes described herein is an acrydite moiety that is covalentlyincorporated into a polyacrylamide matrix, or a primary amine that iscovalently incorporated into a proteinaceous BSPEG matrix. Exemplarymethods of attaching nucleic acids to matrices for purposes ofamplification and/or sequencing are known to those of skill in the artas provided in WO2014/163886.

The disclosure provides that the nucleic acid probes are modified toincorporate a functional moiety for attachment to the matrix. Thefunctional moiety can be covalently cross-linked, copolymerize with orotherwise non-covalently bound to the matrix. The functional moiety canreact with a cross-linker. The functional moiety can be part of aligand-ligand binding pair. dNTP or dUTP can be modified with thefunctional group, so that the function moiety is introduced into the DNAduring amplification. A suitable exemplary functional moiety includes anamine, acrydite, alkyne, biotin, azide, and thiol. In the case ofcrosslinking, the functional moiety is cross-linked to modified dNTP ordUTP or both. Suitable exemplary cross-linker reactive groups includeimidoester (DMP), succinimide ester (NHS), maleimide (Sulfo-SMCC),carbodiimide (DCC, EDC) and phenyl azide. Cross-linkers within the scopeof the present disclosure may include a spacer moiety. Such spacermoieties may be functionalized. Such spacer moieties may be chemicallystable. Such spacer moieties may be of sufficient length to allowamplification of the nucleic acid bound to the matrix. Suitableexemplary spacer moieties include polyethylene glycol, carbon spacers,photo-cleavable spacers and other spacers known to those of skill in theart and the like. Matrix forming materials include polyacrylamide,cellulose, alginate, polyamide, cross-linked agarose, cross-linkeddextran or cross-linked polyethylene glycol. The matrix formingmaterials can form a matrix by polymerization and/or crosslinking of thematrix forming materials using methods specific for the matrix formingmaterials and methods, reagents and conditions known to those of skillin the art.

Hybridization of Probes to a Target Nucleic Acid Sequence

Hybridization of the probes of the invention to target chromosomessequences can be accomplished by standard in situ hybridization (ISH)techniques (see, e.g., Gall and Pardue (1981) Meth. Enzymol. 21:470;Henderson (1982) Int. Review of Cytology 76:1). Generally, ISH comprisesthe following major steps: (1) fixation of the biological structure tobe analyzed (e.g., a chromosome spread), (2) pre-hybridization treatmentof the biological structure to increase accessibility of target DNA(e.g., denaturation with heat or alkali), (3) optional pre-hybridizationtreatment to reduce nonspecific binding (e.g., by blocking thehybridization capacity of repetitive sequences), (4) hybridization ofthe mixture of nucleic acids to the nucleic acid in the biologicalstructure or tissue; (5) post-hybridization washes to remove nucleicacid fragments not bound in the hybridization and (6) detection of thehybridized labelled oligonucleotides (e.g., hybridized Oligopaints). Thereagents used in each of these steps and their conditions of use varydepending on the particular situation. For instance, step 3 will notalways be necessary as the probes described herein can be designed toavoid repetitive sequences. Hybridization conditions are also describedin U.S. Pat. No. 5,447,841. It will be appreciated that numerousvariations of in situ hybridization protocols and conditions are knownand may be used in conjunction with the present invention bypractitioners following the guidance provided herein.

As used herein, the term “hybridization” refers to the process in whichtwo single-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide. The term “hybridization” may also referto triple-stranded hybridization. The resulting (usually)double-stranded polynucleotide is a “hybrid” or “duplex.”Oligonucleotide probes according to the present disclosure need not forma perfectly matched duplex with the single stranded nucleic acid, thougha perfect matched duplex is exemplary. According to one aspect,oligonucleotide probes as described herein form a stable hybrid withthat of the target sequence under stringent to moderately stringenthybridization and wash conditions. If it is expected that the probeswill be essentially completely complementary (i.e., about 99% orgreater) to the target sequence, stringent conditions will be used. Ifsome mismatching is expected, with the result that the probe will not becompletely complementary, the stringency of hybridization may belessened. “Hybridization conditions” will typically include saltconcentrations of less than about 1 M, more usually less than about 500mM and even more usually less than about 200 mM. Hybridizationtemperatures can be as low as 5° C., but are typically greater than 22°C., more typically greater than about 30° C., and often in excess ofabout 37° C. Hybridizations are usually performed under stringentconditions, i.e., conditions under which a probe will hybridize to itstarget subsequence. Stringent conditions are sequence-dependent and aredifferent in different circumstances. Longer fragments may requirehigher hybridization temperatures for specific hybridization. As otherfactors may affect the stringency of hybridization, including basecomposition and length of the complementary strands, presence of organicsolvents and extent of base mismatching, the combination of parametersis more important than the absolute measure of any one alone. Generally,stringent conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH. Exemplarystringent conditions include salt concentration of at least 0.01 M to nomore than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3and a temperature of at least 25° C. For example, conditions of 5×SSPE(750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperatureof 25-30° C. are suitable for allele-specific probe hybridizations. Forstringent conditions, see for example, Sambrook, Fritsche and Maniatis,Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press(1989) and Anderson Nucleic Acid Hybridization, 1^(st) Ed., BIOSScientific Publishers Limited (1999). “Hybridizing specifically to” or“specifically hybridizing to” or like expressions refer to the binding,duplexing, or hybridizing of a molecule substantially to or only to aparticular nucleotide sequence or sequences under stringent conditionswhen that sequence is present in a complex mixture (e.g., totalcellular) DNA or RNA. It is to be understood that any desired stringencyand/or conditions may be employed as desired.

Detection method(s) used will depend on the particularfluorescence-based sequencing methods used to sequence the barcodes ofthe Oligopaints. In certain exemplary embodiments, chromosomes and/orchromosomal regions having one or more Oligopaints bound thereto andhaving fluorescent moieties associated therewith because of thefluorescence-based sequencing method may be detected using a microscope,a spectrophotometer, a tube luminometer or plate luminometer, x-rayfilm, a scintillator, a fluorescence activated cell sorting (FACS)apparatus, a microfluidics apparatus or the like. Detection may beaccomplished using detection devices commonly use with FISSEQ methodsknown to those of skill in the art.

The in situ hybridization methods described herein can be performed on avariety of biological or clinical samples, in cells that are in any (orall) stage(s) of the cell cycle (e.g., mitosis, meiosis, interphase, G0,G1, S and/or G2). Examples include all types of cell culture, animal orplant tissue, peripheral blood lymphocytes, buccal smears, touchpreparations prepared from uncultured primary tumors, cancer cells, bonemarrow, cells obtained from biopsy or cells in bodily fluids (e.g.,blood, urine, sputum and the like), cells from amniotic fluid, cellsfrom maternal blood (e.g., fetal cells), cells from testis and ovary,and the like. Samples are prepared for assays of the invention usingconventional techniques, which typically depend on the source from whicha sample or specimen is taken. These examples are not to be construed aslimiting the sample types applicable to the methods and/or compositionsdescribed herein.

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures, tables, andaccompanying claims.

Nucleic Acid

The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acidsequence,” “nucleic acid fragment,” “oligonucleotide” and“polynucleotide” are used interchangeably and are intended to include,but not limited to, a polymeric form of nucleotides that may havevarious lengths, either deoxyribonucleotides or ribonucleotides, oranalogs thereof. Different polynucleotides may have differentthree-dimensional structures, and may perform various functions, knownor unknown. Non-limiting examples of polynucleotides include a gene, agene fragment, an exon, an intron, intergenic DNA (including, withoutlimitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA,ribosomal RNA, ribozymes, small interfering RNA (siRNA), cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of a sequence, isolated RNA of a sequence, nucleicacid probes, and primers. Oligonucleotides or polynucleotides useful inthe methods described herein may comprise natural nucleic acid sequencesand variants thereof, artificial nucleic acid sequences, or acombination of such sequences. Oligonucleotides or polynucleotides maybe single stranded or double stranded.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

Examples of modified nucleotides include, but are not limited todiaminopurine, S²T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,2,6-diaminopurine and the like. Nucleic acid molecules may also bemodified at the base moiety (e.g., at one or more atoms that typicallyare available to form a hydrogen bond with a complementary nucleotideand/or at one or more atoms that are not typically capable of forming ahydrogen bond with a complementary nucleotide), sugar moiety orphosphate backbone. Nucleic acid molecules may also containamine-modified groups, such as aminoallyl-dUTP (aa-dUTP) andaminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment ofamine reactive moieties, such as N-hydroxy succinimide esters (NHS).

Oligonucleotide sequences, such as single stranded oligonucleotidesequences, may be isolated from natural sources, synthesized orpurchased from commercial sources. In certain exemplary embodiments,oligonucleotide sequences may be prepared using one or more of thephosphoramidite linkers and/or sequencing by ligation methods known tothose of skill in the art. Oligonucleotide sequences may also beprepared by any suitable method, e.g., standard phosphoramidite methodssuch as those described herein below as well as those described byBeaucage and Carruthers ((1981) Tetrahedron Lett. 22: 1859) or thetriester method according to Matteucci et al. (1981) J. Am. Chem. Soc.103:3185), or by other chemical methods using either a commercialautomated oligonucleotide synthesizer or high-throughput, high-densityarray methods known in the art (see U.S. Pat. Nos. 5,602,244, 5,574,146,5,554,744, 5,428,148, 5,264,566, 5,141,813, 5,959,463, 4,861,571 and4,659,774, incorporated herein by reference in its entirety for allpurposes). Pre-synthesized oligonucleotides may also be obtainedcommercially from a variety of vendors.

In certain exemplary embodiments, oligonucleotide sequences may beprepared using a variety of microarray technologies known in the art.Pre-synthesized oligonucleotide and/or polynucleotide sequences may beattached to a support or synthesized in situ using light-directedmethods, flow channel and spotting methods, inkjet methods, pin-basedmethods and bead-based methods set forth in the following references:McGall et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:13555; SyntheticDNA Arrays In Genetic Engineering, Vol. 20:111, Plenum Press (1998);Duggan et al. (1999) Nat. Genet. S21:10; Microarrays: Making Them andUsing Them In Microarray Bioinformatics, Cambridge University Press,2003; U.S. Patent Application Publication Nos. 2003/0068633 and2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890, 6,824,866, 6,800,439,6,375,903 and 5,700,637; and PCT Application Nos. WO 04/031399, WO04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO 03/065038, WO03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO 03/040410 and WO02/24597.

Polymerase recognition sites, cleavage sites and/or label or detectablemoiety addition sites may be added to the single strandedoligonucleotides during synthesis using known materials and methods.

Solid Phase Supports or Substrates

In certain exemplary embodiments, one or more template nucleic acidsequences, i.e. oligonucleotide sequences, described herein areimmobilized on a support or substrate made of materials known to thoseof skill in the art such glass or polymeric materials (e.g., a solidand/or semi-solid support). In certain aspects, an oligonucleotidesequence can be attached to a support using one or more of thephosphoramidite linkers described herein. Suitable supports include, butare not limited to, slides, beads, chips, particles, strands, gels,sheets, tubing, spheres, containers, capillaries, pads, slices, films,plates and the like. In various embodiments, a solid support may bebiological, nonbiological, organic, inorganic, or any combinationthereof. When using a support that is substantially planar, the supportmay be physically separated into regions, for example, with trenches,grooves, wells, or chemical barriers (e.g., hydrophobic coatings, etc.).

In certain exemplary embodiments, a support is a microarray. As usedherein, the term “microarray” refers in one embodiment to a type ofassay that comprises a solid phase support having a substantially planarsurface on which there is an array of spatially defined nonoverlappingregions or sites that each contain an immobilized nucleic acid such as ahybridization probe. “Substantially planar” means that features orobjects of interest, such as probe sites, on a surface may occupy avolume that extends above or below a surface and whose dimensions aresmall relative to the dimensions of the surface. For example, beadsdisposed on the face of a fiber optic bundle create a substantiallyplanar surface of probe sites, or oligonucleotides disposed orsynthesized on a porous planar substrate create a substantially planarsurface. Spatially defined sites may additionally be “addressable” inthat its location and the identity of the immobilized probe at thatlocation are known or determinable.

Oligonucleotides immobilized on microarrays include nucleic acids thatare generated in or from an assay reaction. Typically, theoligonucleotides or polynucleotides on microarrays are single strandedand are covalently attached to the solid phase support, usually by a5′-end or a 3′-end. In certain exemplary embodiments, oligonucleotidesare immobilized via one or more of the cleavable linkers describedherein. One or more or a plurality of cleavable moieties may also belocated internally within the oligonucleotides, thereby providing sitesto cleave a rolling circle amplification product into smaller nucleicacid sequences. The density of nonoverlapping regions containing nucleicacids in a microarray is typically greater than 100 per cm², and moretypically, greater than 1000 per cm². Microarray technology relating tonucleic acid sequences is reviewed in the following exemplaryreferences: Schena, Editor, Microarrays: A Practical Approach (IRLPress, Oxford, 2000); Southern, Current Opin. Chem. Biol., 2: 404-410(1998); Nature Genetics Supplement, 21:1-60 (1999); and Fodor et al,U.S. Pat. Nos. 5,424,186; 5,445,934; and 5,744,305. Oligonucleotidesbound to a solid support useful in the present methods are commerciallyavailable and can be designed and made using methods known to those ofskill in the art.

Methods of immobilizing oligonucleotides to a support are known in theart (beads: Dressman et al. (2003) Proc. Natl. Acad. Sci. USA 100:8817,Brenner et al. (2000) Nat. Biotech. 18:630, Albretsen et al. (1990)Anal. Biochem. 189:40, and Lang et al. Nucleic Acids Res. (1988)16:10861; nitrocellulose: Ranki et al. (1983) Gene 21:77; cellulose:Goldkorn (1986) Nucleic Acids Res. 14:9171; polystyrene: Ruth et al.(1987) Conference of Therapeutic and Diagnostic Applications ofSynthetic Nucleic Acids, Cambridge U.K.; teflon-acrylamide: Duncan etal. (1988) Anal. Biochem. 169:104; polypropylene: Polsky-Cynkin et al.(1985) Clin. Chem. 31:1438; nylon: Van Ness et al. (1991) Nucleic AcidsRes. 19:3345; agarose: Polsky-Cynkin et al., Clin. Chem. (1985) 31:1438;and sephacryl: Langdale et al. (1985) Gene 36:201; latex: Wolf et al.(1987) Nucleic Acids Res. 15:2911).

As used herein, the term “attach” refers to both covalent interactionsand noncovalent interactions. A covalent interaction is a chemicallinkage between two atoms or radicals formed by the sharing of a pair ofelectrons (i.e., a single bond), two pairs of electrons (i.e., a doublebond) or three pairs of electrons (i.e., a triple bond). Covalentinteractions are also known in the art as electron pair interactions orelectron pair bonds. Noncovalent interactions include, but are notlimited to, van der Waals interactions, hydrogen bonds, weak chemicalbonds (i.e., via short-range noncovalent forces), hydrophobicinteractions, ionic bonds and the like. A review of noncovalentinteractions can be found in Alberts et al., in Molecular Biology of theCell, 3d edition, Garland Publishing, 1994.

Example I

FIG. 1 depicts a DNA oligonucleotide probe, referred to here as an“oligopaint,” is designed to contain a region for hybridization to thegenome (complementary region or sequence), as well as twobarcode-containing regions flanking the complementary region. It is tobe understood that only a single barcode region on either side of thecomplementary region, i.e. 5′ or 3′ side, can be used. A plurality ofprobes is hybridized to the genome indicated in red and blue. Multipleprobes complementary to a contiguous region of the genome indicated inred or blue bear a single unique associated barcode. It is to beunderstood that any number of contiguous regions of the genome can betargeted with probes having a unique associated barcode for a particularcontiguous region. The barcodes are used for fluorescent identificationof the genomic locus inside fixed cells or along the condensed chromatinof a metaphase spread.

FIG. 2 depicts that the DNA oligonucleotide probe, referred to here asan “oligopaint,” is designed to incorporate a barcode-containing regioncompatible with sequencing by ligation, sequencing by synthesis, orsequencing by hybridization. Information contained in the barcode isread out fluorescently to generate a barcode, which can becomputationally mapped to a genomic locus based on the design of theprobe library. Here two genomic loci, each 25 kilobases in length, aregiven distinct barcodes and detected using a number of probes. Using alarge number of probes to barcode a single genomic locus increases thephoton yield and signal-to-noise ratio for fluorescent microscopicdetection.

FIG. 3 depicts that a genomic locus is targeted by a plurality ofoligopaint probes, each bearing a subset of the identifying barcode forthe genomic locus. For each oligopaint, regions bearing complementarityto the genomic locus, which drive targeted hybridization to the genomiclocus, are in blue. Barcode encoding regions are in red, spacers are inblack. To detect the first segment of the barcode, four linkers arehybridized to the sample, followed by HCR to generate an amplifiedfluorescent signal in one of four spectrally distinct colors. At thislocus, the ID “1” is determined at the first segment of the barcode. TheHCR is reversed and the linker is cleaved as in cyclic reversible HCR. Asecond round of linker hybridization and HCR occurs, identifying ID 0 asthe second segment of the barcode. This process is repeated until acomplete barcode is recovered.

FIG. 4 depicts a pool of oligopaints hybridized against a metaphasespread genome (left) and an intact nucleus (right). The oligopaint poolis designed such that a subset of the pool is targeted to a single siteon the q arm of each chromosome, approximately 100 kb in size. (Hundredsof probes are targeted to each site). All oligopaints for each locusbear a distinct barcode designed to be detected by sequencing byligation (SBL). A sequencing probe is hybridized to the oligopaints, andSBL is used to detect the first segment, in this case the first base, ofa nucleic acid barcode sequence. Shown is a 3 color image of a 4 colorsequencing reaction. After sequencing 5 bases with SBL, each barcode isidentified, such that the target locus is labeled and detected in situ.

Example II Oligopaints are Sequenced Using FISSEQ within an ExpandedMatrix

PGP1F cells were plated onto glass microscope slides and were allowed toadhere overnight at 37° C. in a cell culture incubator. On the next day,cells on slides were transferred to coplin jars and washed 1× with1×PBS, followed by fixation with 4% formaldehyde in PBS for 10 min at RT(room temperature). Another 1×PBS wash was performed and cells werepermeabalized in 1×PBS+0.5% Triton X-100 for 10 min at RT. Two1×PBS+0.1% Triton X-100 (1×PBT) washes for 5 minutes were performed atRT for 5 min each. Slides could then be stored in 1×PBT at 4° C. or beused for experiments. If proceeding to experiments, slides were treatedin 0.1N HCl for 5 min at RT. Two washes in 2×SSCTween for 5 min at RTwere performed. Next, pre-hybridization was done in 50% formamide in2×SSCT solution for 5 min at RT. Another wash in same buffer was done at60° C. for 20 min. Slides were slightly air-dried and 25 uL of probesolution including 100 pmols of Oligopaints in 50% formamide, 10%Polyacrylic Acid, 2×SSCT, and 20 mg RNAse1 were added to each slide,covered with a 22×22 mm cover slip and sealed with rubber cement. Probeswere hybridized 0/N at 42° C. in a humid chamber. The next day, rubbercement and coverslips were carefully removed. Unbound probes were washedoff with 2×SSCT at 60° C. for 20 min. 2×2×SSCT washes were performed atRT for 5 min each, followed by 0.2×SSC wash for 5 min at RT. 30 uL ofExM gel was cast over cells on slide using gelation chamber (parafilmwrapped glass microscope slide with 2 22×22 1.5 coverslips used asspacers) and allowed to polymerize at 37° C. for 1 hr. Afterpolymerization, gelation chamber was carefully removed and gels on slidewere digested 0/N in digestion buffer and 1:100 of NEB Proteinase K (20mg/mL) at 37° C. Following digestion, glass slides were removed and gelswere expanded in 1×PBS by shaking 2× for 7 min at RT. To ensure thatgels remained expanded during subsequent steps, gels were re-embedded.Gels were tilted in 1.5 mL tube with 3% Acrylamide/BIS in 1×PBS with0.05% APS and 0.05% TEMED for 20 min at RT. Gels were then removed andplaced on microscope slide. A piece of 1.5 coverslip, broken to be bigenough to cover the gel, was placed over the gel. Covered gels onmicroscope slide were placed in humid chamber that was filled with Argongas to remove oxygen from chamber. Gelling proceeded at 37° C. for 1 hr.Re-embedded gels were washed 1× for 7 min in 100 mM MES at RT. Sampleswere passivated for 2 hrs at RT in 150 mM EDC, 150 mM NHS, 2MEthanolamine hydrochloride, and 5M NaCl. Ethanolamine was then reactedfor 40 min at RT by adding 2M Ethanolamine hydrochloride, 62.5 mM SodiumBorate Buffer (pH 8.5), and 5M NaCl to gels. Gels were then washed 3× in1× SoLiD Instrument Buffer for 10 min at RT. Wash in 1×T4 Ligase Bufferfor 7 min at RT was done to prepare for Oligopaint circularization.Oligopaints were then circularized by adding 2 uM Oligo Splint in 1×T4Ligase Buffer and T4 DNA Ligase for 2 hrs at RT with gentle shaking.Samples were then washed 2× in 1× Instrument buffer for 7 min at RT,followed by washing in 1×NEB Buffer 1 for 7 min at RT. ssDNA andnon-circularized Oligopaints were degraded by 1 uL NEB Exonuclease I in1× Exonuclease I Buffer for 45 min at 37° C. 3×1× instrument bufferwashes for 7 min at RT, then 1×10 min wash in 30% formamide in 2×SSC atRT. Hybridization of 1 uM Rolling Circle Amplification (RCA) primer(same as Splint) was done in 30% formamide and 2×SSC for 1 hr at RT.After RCA primer hybridization, samples were washed 2×10 min ininstrument buffer followed by 1×10 min in Phi29 Polymerase Buffer. RCAwas performed by adding 1×Phi29 buffer, 250 uM dNTPs, 20 uM aminoallyldUTP, and 2 Units of Phi29 DNA polymerase 0/N at 30° C. RCA ampliconswere crosslinked for 30 min at RT in 20 uL BS(PEG)9 in 980 uL 1×PBS.Quenching of BS(PEG)9 was done by incubating samples in 1M Tris (pH 8.0)for 45 min. Quenched samples were then washed 3×10 min in 1× instrumentbuffer at RT. To prepare for sequencing, 2.5 uM sequencing primer in5×SSCT was hybridized for 1 hr at RT. 2×10 min washes in 1× instrumentbuffer was done, followed by washing in 1×T4 DNA ligase buffer for 10min at RT. To sequence, 1×T4 ligation buffer, 5 uL of T4 DNA ligase, 1uL of SoLiD sequencing nucleotide mix, with 84 uL of water was added toeach sample for 2 hrs at RT. Samples were then washed with 1× instrumentbuffer for 1 hr at RT before imaging. FIG. 5 shows that Oligopaintcircularization, followed by Rolling Circle Amplification, and 1 roundof SoLiD sequencing by ligation is achieved in-situ, in fixed andexpanded human cells.

Example III Oligopaints with an Acrydite Attachment Moiety are Attachedto a Matrix and Hybridized with a Secondary Oligo Bearing a DetectableMoiety

Oligopaint library was first PCR amplified linearly to limit PCR errors.PCR product was column purified and resuspended in water. This linearproduct was PCR amplified again and T7 promoter sequences were added toeach backstreet (non-genomic sequence downstream of the complementarysequence) via reverse primer. 1.3 ug of purified PCR products werein-vitro transcribed O/N at 37° C. This RNA served as the template forreverse transcription using forward primers containing 5′ Acryditemodification to append Acrydite to Oligopaint. RNA was degraded byalkaline hydrolysis. Samples were then purified with Zymo 100 columnpurification kit, using Oligobinding Buffer instead of DNA bindingbuffer. Acrydite modified oligopaints were then used in standard FISHprotocol. Following 0/N hybridization of Acrydite modified Oligopaints,ExM (expandable matrix) gel was cast over samples using gelation chamberand allowed to polymerize at 37° C. for 1 hr. Gels were digested 0/N indigestion buffer with 1:100 Proteinase K (NEB). Gels were washed 3×30min in 2×SSCT. Gels were then cut and separated such that some samplescould be probed with secondary oligo (Sample N) and some could bedenatured prior to probing with secondary probe (Sample D) to assesstethering to ExM gel matrix. Sample N were kept in 2×SSCT at RT. SampleD were incubated in 70% formamide/2×SSC at 73° C. for 3 min 2× toconfirm tethering of Acrydite modified Oligopaint to matrix. Sample Dwas then washed with 2×SSCT 2× for 10 min at RT. 3.3 uM secondary oligoswere hybridized to Sample N and D for 1 hr at RT. Gels were washed in30% formamide in 2×SSCT for 2× at RT for 30 min. 2 washes in 2×SSCT for10 min were performed, followed by 1:500 DAPI in PBS staining for 20 minat RT. Samples were then imaged. FIG. 6A-6E are directed to thesuccessful synthesis of Oligopaints with an Acrydite modification at the5′ end as the Oligopaints remain tethered and are able to be hybridizedby a secondary oligo after treatment with high concentration offormamide with heating (FIG. 6E). This is in contrast to non-modifiedOligopaints that are no longer present in the sample.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention. Other embodiments will be evident to those ofskill in the art. It should be understood that the foregoing descriptionis provided for clarity only and is merely exemplary. All publications,patents and patent applications cited above are incorporated byreference herein in their entirety for all purposes to the same extentas if each individual publication or patent application werespecifically indicated to be so incorporated by reference.

What is claimed is:
 1. A method of identifying a target genomic nucleicacid sequence comprising hybridizing a set of probes to the targetgenomic nucleic acid sequence, wherein the set of probes has a uniqueassociated barcode sequence for identification of the target genomicnucleic acid sequence, wherein each probe of the set includes (1) acomplementary sequence complementary to a first strand or a secondstrand of the target genomic nucleic acid sequence and (2) theassociated barcode sequence or a portion of the associated barcodesequence, sequencing the associated barcode sequence from probeshybridized to the target genomic nucleic acid sequence using afluorescence-based sequencing method, and identifying the target genomicnucleic acid sequence by the sequenced barcode sequence.
 2. The methodof claim 1 wherein the fluorescence-based sequencing method issequencing by synthesis and the probe further includes a primingsequence for annealing a sequencing primer, wherein the sequencingprimer is extended by a DNA polymerase using reversible terminatorfluorescently encoded dNTPs to generate a fluorescent signalcorresponding to the barcode.
 3. The method of claim 1 wherein thefluorescence-based sequencing method is sequencing by ligation and theprobe further includes a priming sequence for annealing a sequencingprimer, wherein the sequencing primer is extended in either the 5′ or 3′direction by a DNA ligase using fluorescently encoded oligonucleotidesto generate a fluorescent signal corresponding to the barcode.
 4. Themethod of claim 1 wherein the fluorescence-based sequencing method issequencing by hybridization and the probe further includes a firstnucleic acid sequence complementary to a labeled oligonucleotide whichhybridizes to the first nucleic acid sequence.
 5. The method of claim 1wherein the fluorescence-based sequencing method is sequencing by cyclicreversible polymerization hybridization chain reaction.
 6. The method ofclaim 1 wherein a plurality of probes having a portion of the associatedbarcode sequence constitute a complete associated barcode sequence. 7.The method of claim 1 wherein the complementary sequence has anucleotide length of between 5 and 10,000 bases.
 8. The method of claim1 wherein the complementary sequence has a nucleotide length of between15 and 1,000 bases.
 9. The method of claim 1 wherein the complementarysequence has a nucleotide length of between 20 and 80 bases.
 10. Themethod of claim 1 wherein the probe includes one or more spacersequences separating the complementary sequence from the associatedbarcode sequence or portion of the associate barcode sequence.
 11. Themethod of claim 1 wherein the probe includes one or more spacersequences comprising a plurality of dT nucleotides separating thecomplementary sequence from the associated barcode sequence or portionof the associate barcode sequence.
 12. The method of claim 1 wherein theprobe includes one or more spacer sequences separating functionalsequences of the barcode including the complementary sequence, theassociated barcode sequence or portion of the associate barcodesequence, and a priming sequence.
 13. The method of claim 1 wherein theprobe includes one or more spacer sequences comprising a plurality of dTnucleotides separating functional sequences of the barcode including thecomplementary sequence, the associated barcode sequence or portion ofthe associate barcode sequence, and a priming sequence.
 14. The methodof claim 1 wherein the target genomic nucleic acid has a length ofbetween 10 bp and 1,000,000,000 bp.
 15. The method of claim 1 whereinthe target genomic nucleic acid has a length of between 20 bp and1,000,000 bp.
 16. The method of claim 1 wherein the target genomicnucleic acid has a length of between 100 bp and 1,000,000 bp.
 17. Themethod of claim 1 wherein the target genomic nucleic acid has a lengthof between 100,000 bp and 1,000,000,000 bp.
 18. The method of claim 1wherein the target genomic nucleic acid is a whole genome.
 19. Themethod of claim 1 wherein the probe includes one or more additionalbarcode sequences for barcoding characteristics of the target genomicnucleic acid.
 20. The method of claim 1 wherein the probes areoligopaints.
 21. The method of claim 1 wherein the probes furtherinclude an attachment moiety for attachment to a matrix.
 22. A method ofmultiplexing the identification of a plurality of target genomic nucleicacid sequences within genomic DNA comprising hybridizing the genomic DNAwith a plurality of probe sets corresponding to the plurality of targetgenomic nucleic acid sequences, wherein each probe set has a uniqueassociated barcode sequence for identification of the correspondingtarget genomic nucleic acid sequence, wherein each probe of each probeset includes (1) a complementary sequence complementary to a firststrand of the corresponding target genomic nucleic acid sequence and (2)the associated barcode sequence or a portion of the associated barcodesequence, sequencing the associated barcode sequence from probeshybridized to the plurality of target genomic nucleic acid sequencesusing a fluorescence-based sequencing method, and identifying the targetgenomic nucleic acid sequence by the sequenced barcode sequence.
 23. Themethod of claim 22 wherein the plurality of target genomic nucleic acidsequences within genomic DNA is between 5 and 100,000,000 target genomicnucleic acid sequences.